Polynucleotide matrix-based method of identifying microorganisms

Abstract

Method of identifying microbial organisms wherein a biological sample containing nucleic acids is hybridized with a collection of polynucleotide probes, each probe having binding specificity for the ribosomal nucleic acids of at least one microbe. The collection of probes is organized into a series of "addresses" that provide information about the presence or absence of one or more polynucleotide sequences in the biological sample. Probes in the matrix are selected to distinguish between organisms that differ from each other by a known phylogenetic relationship. Advantageously, the invented method can detect and resolve the identities of microorganisms that are present in a mixed culture. The system is particularly suited to automated analysis.

What is claimed is:  
     
       1. A device for detecting nucleic acids, comprising 
       a solid support; and  
       a plurality of addresses disposed on the solid support, each address comprising at least one detectably labeled probe that hybridizes ribosomal nucleic acids from at least one microbial species under high stringency hybridization conditions, said plurality of addresses comprising,  
       a pan-bacterial address,  
       a higher-order address,  
       wherein said higher-order address is a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species,  
       an intermediate-order address, and  
       a lower-order address,  
       wherein the lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address,  
       wherein the intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address, and  
       wherein at least one of said intermediate-order address and said lower-order address further comprise a detectably labeled probe that hybridizes ribosomal nucleic acids from a microbial species having ribosomal nucleic acids that hybridize at the pan-bacterial address.  
     
     
       2. A device for detecting nucleic acids, comprising: 
       a solid support; and  
       a plurality of addresses disposed on the solid support, each address comprising at least one detectably labeled probe that hybridizes ribosomal nucleic acids from at least one microbial species under high stringency hybridization conditions, said plurality of addresses comprising,  
       a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species;  
       a higher-order address,  
       wherein said higher-order address is a pan-bacterial address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram (+)  bacteria, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus  Enterococcus , a plurality of species of bacteria in the genus  Staphylococcus , and a plurality of species of bacteria in the genus  Campylobacter,    
       a first intermediate-order address, and  
       a first lower-order address,  
       wherein the first lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the first intermediate-order address,  
       wherein the first intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address, and  
       wherein said first lower-order address further comprises a detectably labeled probe that  
       hybridizes ribosomal nucleic acids from a microbial species having ribosomal nucleic acids that hybridize at the higher-order address  
       but does not hybridize ribosomal nucleic acids from any microbial species having ribosomal nucleic acids that hybridize at said first intermediate-order address.  
     
     
       3. The device of either  claim 1  or  claim 2 , wherein the solid support is selected from the group consisting of a multiwell plate, and a plurality of individual tubes each maintained in a spaced-apart configuration. 
     
     
       4. The device of  claim 1 , wherein the intermediate-order address specifically hybridizes ribosomal nucleic acids from a plurality of  Candida  species comprising  Candida albicans, Candida tropicalis, Candida dubliniensis, Candida viswanathii  and  Candida parapsilosis , and wherein the lower-order address specifically hybridizes ribosomal nucleic acids from  Candida albicans  and  Candida dubliniensis  but not  Candida tropicalis, Candida viswanathii  or  Candida parapsilosis.    
     
     
       5. The device of  claim 1  or  claim 2 , wherein each of said at least one detectably labeled probe is labeled with an acridinium ester. 
     
     
       6. The device of  claim 1 , wherein each of said at least one detectably labeled probes is at least one detectably labeled soluble probe. 
     
     
       7. The device of  claim 1 , wherein said intermediate-order address specifically hybridizes ribosomal nucleic acids of  Candida  species comprising  Candida albicans  and  Candida dubliniensis.    
     
     
       8. The device of  claim 7 , wherein said lower-order address specifically hybridizes ribosomal nucleic acids of  Candida albicans.    
     
     
       9. The device of  claim 1 , wherein said intermediate-order address specifically hybridizes ribosomal nucleic acids of  Candida  species comprising  Candida albicans, Candida dubliniensis, Candida tropicalis, Candida viswanathii  and  Candida parapsilosis.    
     
     
       10. The device of  claim 9 , wherein said lower-order address specifically hybridizes ribosomal nucleic acids of  Candida albicans.    
     
     
       11. The device of  claim 2 , wherein said first intermediate-order address is selected from the group consisting of a Gram (+)  address that specifically hybridizes ribosomal nucleic acids of a plurality of Gram (+)  bacteria, wherein said plurality of Gram (+)  bacteria comprise a plurality of bacteria in the genus  Staphylococcus , a plurality of bacteria in the genus  Enterococcus , and a plurality of  Actinomycetes  bacteria in the High(G+C) subset of Gram (+)  bacteria, a family Enterobacteriaceae address that specifically hybridizes ribosomal nucleic acids from a plurality of bacteria in the family Enterobacteriaceae, a  Staphylococcus  genus address that specifically hybridizes ribosomal nucleic acids from a plurality of species in the  Staphylococcus  genus, a genus  Enterococcus  address that specifically hybridizes ribosomal nucleic acids from a plurality of species in the genus  Enterococcus , and a  Campylobacter  address that specifically hybridizes ribosomal nucleic acids from a plurality of  Campylobacter  species. 
     
     
       12. The device of  claim 11 , wherein the first intermediate-order address is the Gram (+)  address, and wherein the first lower-order address is an address that specifically hybridizes ribosomal nucleic acids from a plurality of  Mycobacterium  species. 
     
     
       13. The device of  claim 12 , wherein said plurality of  Mycobacterium  species comprises  Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis  BCG and  Mycobacterium africanum.    
     
     
       14. The device of  claim 11 , wherein the first intermediate-order address is the Gram (+)  address, and wherein the first lower-order address is an address that specifically hybridizes ribosomal nucleic acids of  Streptococcus pneumoniae.    
     
     
       15. The device of  claim 11 , wherein the first intermediate-order address is the Gram (+)  address, and wherein the first lower-order address is an address that specifically hybridizes ribosomal nucleic acids from  Listeria monocytogenes.    
     
     
       16. The device of  claim 11 , wherein the first intermediate-order address is the Gram (+)  address, and wherein the first lower-order address is an address that specifically hybridizes ribosomal nucleic acids of  Staphylococcus aureus.    
     
     
       17. The device of  claim 11 , wherein the first intermediate-order address is the family Enterobacteriaceae address, and wherein the first lower-order address is an  E. coli  address that specifically hybridizes ribosomal nucleic acids of  E. coli.    
     
     
       18. The device of  claim 11 , wherein the first intermediate-order address is the  Staphylococcus  genus address, and wherein the first lower-order address is a  Staphylococcus aureus  address that specifically hybridizes ribosomal nucleic acids from  Staphylococcus aureus.    
     
     
       19. The device of  claim 11 , wherein the first intermediate-order address is the genus  Enterococcus  address. 
     
     
       20. The device of  claim 11 , wherein the first intermediate-order address is the  Campylobacter  address. 
     
     
       21. The device of  claim 11 , wherein the first intermediate-order address is the Gram (+)  address, and wherein the first lower-order address is an  Actinomycetes  address that specifically hybridizes ribosomal nucleic acids of a plurality of bacteria belonging to the High (G+C) subset of Gram (+)  bacteria, wherein said plurality of bacteria belonging to the High(G+C) subset of Gram (+)  bacteria comprise a plurality of bacteria in the genus  Corynebacterium  and a plurality of bacteria in the genus  Mycobacterium.    
     
     
       22. The device of  claim 21 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from bacteria in the family Enterobacteriaceae. 
     
     
       23. The device of  claim 22 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from  Enterococcus  bacteria. 
     
     
       24. The device of  claim 23 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from bacteria in the  Staphylococcus  genus and an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus  Campylobacter.    
     
     
       25. The device of  claim 24 , wherein said plurality of addresses further includes at least one address that specifically hybridizes ribosomal nucleic acids from a single microorganism species. 
     
     
       26. The device of  claim 25 , wherein said single microorganism species is selected from the group consisting of  Escherichia coli, Streptococcus pneumoniae, Pseudomonas aeruginosa, Candida albicans  and  Staphylococcus aureus.    
     
     
       27. The device of  claim 21 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from  Enterococcus  bacteria. 
     
     
       28. The device of  claim 21 , further comprising a single address that hybridizes ribosomal nucleic acids of  Enterococcus  bacteria and ribosomal nucleic acids from bacteria in the family Enterobacteriaceae. 
     
     
       29. The device of  claim 28 , wherein said plurality of addresses further includes a single address that hybridizes ribosomal nucleic acids from bacteria in the  Staphylococcus  genus and ribosomal nucleic acids from a plurality of bacteria in the genus  Campylobacter.    
     
     
       30. The device of  claim 29 , wherein said plurality of addresses further includes a plurality of addresses that individually hybridize ribosomal nucleic acids from a plurality of microorganism species. 
     
     
       31. The device of  claim 21 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from bacteria in the  Staphylococcus  genus. 
     
     
       32. The device of  claim 31 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus  Campylobacter.    
     
     
       33. The device of  claim 21 , wherein said plurality of addresses further includes an address that hybridizes ribosomal nucleic acids from a plurality of bacteria in the genus  Campylobacter.    
     
     
       34. The device of  claim 21 , wherein the pan-bacterial address comprises a polynucleotide probe having a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:58. 
     
     
       35. The device of  claim 21 , wherein the pan-fungal address comprises a polynucleotide probe having the sequence of SEQ ID NO:4. 
     
     
       36. The device of  claim 21 , wherein the Gram (+)  address comprises a polynucleotide probe having the sequence of SEQ ID NO:7. 
     
     
       37. The device of  claim 21 , wherein the  Actinomycetes  address comprises a polynucleotide probe having the sequence of SEQ ID NO: 10. 
     
     
       38. The device of  claim 21 , wherein the pan-bacterial address comprises a polynucleotide probe having a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:58, wherein the pan-fungal address comprises a polynucleotide probe having the sequence of SEQ ID NO:4, wherein the Gram (+)  address comprises a polynucleotide probe having the sequence of SEQ ID NO:7, and wherein the  Actinomycetes  address comprises a polynucleotide probe having the sequence of SEQ ID NO:10. 
     
     
       39. The device of  claim 2 , wherein each of said at least one detectably labeled probes is at least one detectably labeled soluble probe. 
     
     
       40. A device for detecting nucleic acids, comprising: 
       a solid support; and  
       a plurality of addresses disposed on the solid support, each address comprising at least one detectably labeled probe that hybridizes ribosomal nucleic acids from at least one microbial species under high stringency hybridization conditions, said plurality of addresses comprising,  
       a pan-fungal address that specifically hybridizes ribosomal nucleic acids from a plurality of fungal species,  
       a higher-order address,  
       wherein said higher-order address is a pan-bacterial address that specifically hybridizes ribosomal nucleic acids from a plurality of species of Gram (+)  bacteria, a plurality of species of bacteria in the family Enterobacteriaceae, a plurality of species of bacteria in the genus  Enterococcus , a plurality of species of bacteria in the genus  Staphylococcus , and a plurality of species of bacteria in the genus  Campylobacter,    
       an intermediate-order address, and  
       a lower-order address,  
       wherein the lower-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the intermediate-order address,  
       wherein the intermediate-order address hybridizes ribosomal nucleic acids from a subset of organisms having ribosomal nucleic acids that hybridize at the higher-order address, and  
       wherein at least one of said intermediate-order address and said lower-order address further comprise a detectably labeled probe that hybridizes ribosomal nucleic acids from a microbial species having ribosomal nucleic acids that hybridize at the pan-fungal address.